Francisella tularensis is a highly infectious bacterium responsible for tularemia, a disease whose pneumonic form has potentially lethal consequences in humans. Francisella virulence depends on its ability to survive and replicate inside macrophages of the infected host, where the bacterium down modulates the macrophage immune functions. The current model of Francisella intracellular fate is initial enclosure within a phagosome, followed by escape from this phagosome and then replication in the cytoplasm, but the bacterial determinants controlling these individual stages are unknown. We have been using cell biology-, bacterial genetics- and genomics-based approaches to further characterize Francisella intracellular trafficking, identify genes expressed at various stages of the intracellular cycle and assess their role in Francisella virulence. Using models of primary macrophage infection with F. tularensis, we have previously established the intracellular cycle of this pathogen, which involves rapid phagosomal escape (Checroun et al., 2006, PNAS, 103:14578;Chong et al., 2008, Infect. Immun., 76:5488) followed by extensive proliferation in the cytosol and autophagy-mediated reentry into the endocytic compartment at late stages of the cycle (Checroun et al., 2006, PNAS, 103:14578;Wehrly et al., 2009, Cell. Microbiol, 11:1128). In our efforts to understand how host factors modulate the Francisella intracellular cycle, we have shown that IFN&#947;activation of macrophages does not affect the ability of Francisella to disrupt its early phagosome, yet restricts cytosolic growth of the bacterium in a manner that is independent of reactive oxygen or nitrogen species, tryptophan depletion or autophagy (Edwards et al., 2010, Microbiology, 156:327). Additionally, we have established that targeting of Francisella to opsonic receptors via either complement or IgG opsonization, a process relevant to nave or immune hosts, negatively affects phagosomal escape and cytosolic proliferation, demonstrating that non-opsonized phagocytic pathways are more permissive to Francisella survival and proliferation than opsonic uptake processes. In our studies of the molecular mechanisms of Francisella intracellular pathogenesis, we have examined the functions encoded within the Francisella Pathogenicity Island (FPI), a locus required for virulence, and demonstrated in collaboration with Dr Karl Klose (University of Texas at San Antonio) that it encodes a functional Type 6 secretion system required for phagosomal escape (Barker et al., 2009, Mol. Microbiol. 74:1459). We have also examined the contribution of acid phosphatases (Acp) in phagosomal escape of virulent strains, as these proteins have been shown to play such a role in the low virulence subspecies novicida. Using genomic comparisons and systematic deletions of acp genes, we have discovered that most acp genes have been disrupted through genome reduction in virulent strains and the most conserved genes between Francisella subspecies do not play any role in pathogenesis of virulent strains (Child et al, 2010, Infect Immun. 78:59). To identify and characterize Francisella genes that are important for intracellular pathogenesis, we have established the intracellular transcriptome of the prototypical virulent strain Schu S4 of F. tularensis and identified novel Francisella-specific genes required for intracellular proliferation and virulence (Wehrly et al., 2009, Cell. Microbiol, 11:1128). Using genetic and biochemical approaches, we have shown that two proteins encoded by these loci, DipA (FTT0369c) and FlpA (FTT1676), are surface-exposed during the intracellular cycle and required for cytosolic replication. Additionally, mutants in either dipA or flpA conferred high levels of protection of mice against a pulmonary challenge with a virulent strain, suggesting that such mutants have some potential as genetically defined, live vaccine strains against tularemia. A patent application for these stable, genetically defined attenuated strains (No. PCT/US10/25417) has been submitted.